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1.  Contributions to Protection from Streptococcus pneumoniae Infection Using the Monovalent Recombinant Protein Vaccine Candidates PcpA, PhtD, and PlyD1 in an Infant Murine Model during Challenge 
A vaccine consisting of several conserved proteins with different functions directing the pathogenesis of pneumonia and sepsis would be preferred for protection against infection by Streptococcus pneumoniae. Infants will be the major population targeted for next-generation pneumococcal vaccines. Here, we investigated the potential efficacy provided by three recombinant pneumococcal vaccine candidate proteins—pneumococcal histidine triad D (PhtD), detoxified pneumolysin derivative (PlyD1), and pneumococcal choline-binding protein A (PcpA)—for reducing pneumonia and sepsis in an infant mouse vaccine model. We found vaccination with PhtD and PcpA provided high IgG antibody titers after vaccination in infant mice, similar to adult mice comparators. PlyD1-specific total IgG was significantly lower in infant mice, with minimal boosting with the second and third vaccinations. Similar isotypes of IgG for PhtD and PlyD1 were generated in infant compared to adult mice. Although lower total specific IgG to all three proteins was elicited in infant than in adult mice, the infant mice were protected from bacteremic pneumonia and sepsis mortality (PlyD1) and had lower lung bacterial burdens (PcpA and PhtD) after challenge. The observed immune responses coupled with bacterial reductions elicited by each of the monovalent proteins support further testing in human infant clinical trials.
PMCID: PMC4135924  PMID: 24850621
2.  Protein carriers of conjugate vaccines 
Human Vaccines & Immunotherapeutics  2013;9(12):2505-2523.
The immunogenicity of polysaccharides as human vaccines was enhanced by coupling to protein carriers. Conjugation transformed the T cell-independent polysaccharide vaccines of the past to T cell-dependent antigenic vaccines that were much more immunogenic and launched a renaissance in vaccinology. This review discusses the conjugate vaccines for prevention of infections caused by Hemophilus influenzae type b, Streptococcus pneumoniae, and Neisseria meningitidis. Specifically, the characteristics of the proteins used in the construction of the vaccines including CRM, tetanus toxoid, diphtheria toxoid, Neisseria meningitidis outer membrane complex, and Hemophilus influenzae protein D are discussed. The studies that established differences among and key features of conjugate vaccines including immunologic memory induction, reduction of nasopharyngeal colonization and herd immunity, and antibody avidity and avidity maturation are presented. Studies of dose, schedule, response to boosters, of single protein carriers with single and multiple polysaccharides, of multiple protein carriers with multiple polysaccharides and conjugate vaccines administered concurrently with other vaccines are discussed along with undesirable consequences of conjugate vaccines. The clear benefits of conjugate vaccines in improving the protective responses of the immature immune systems of young infants and the senescent immune systems of the elderly have been made clear and opened the way to development of additional vaccines using this technology for future vaccine products.
PMCID: PMC4162048  PMID: 23955057
carrier proteins; conjugate vaccines; CRM197; diphtheria toxoid; Haemophilus influenzae protein D; meningococcal outer membrane protein complex; Streptococcus pneumoniae; tetanus toxoid
3.  Nasopharyngeal Bacterial Interactions in Children 
Emerging Infectious Diseases  2012;18(11):1738-1745.
TOC summary: Pathogen prevalence differs during periods of health and at onset of acute otitis media.
PMCID: PMC3559157  PMID: 23092680
Streptococcus pneumoniae; Haemophilus influenzae; Moraxella catarrhalis; Staphylococcus aureus; bacteria; nasopharyngeal bacterial infections; polymicrobial colonization; bacterial interaction; acute otitis media; children
4.  Non- Protective Responses to Pediatric Vaccines Occur in Children Who are Otitis Prone 
The Pediatric infectious disease journal  2013;32(11):10.1097/INF.0b013e31829e887e.
We recently found that children who experience recurrent otitis media despite individualized care (stringently-defined otitis prone, sOP) do not develop an antibody response to several vaccine candidate protein antigens expressed by Streptococcus pneumonia (Spn) and Haemophilus influenzae (Hi). Here we sought to determine if these same children also failed to develop antibody to routine pediatric vaccinations.
Study Design
140 sera collected from children age 6–24 months were analyzed. sOP (n=34) and age-matched non-sOP (n=34) children were assessed for IgG concentrations to diphtheria toxoid (DT), tetanus toxoid (TT), pertussis toxoid (PT), filamentous hemagglutinin (FHA), pertactin (PRN) (DTaP), polio, hepatitis B, Hi type b capsule (PRP) and Streptococcus pneumoniae (Spn) capsular polysaccharide conjugate vaccine.
IgG protective titers to DT (p=0.006), TT (p<0.0001), PT (p<0.0001), FHA (p=0.001), PRN (p=0.005), hepatitis B (p<0.0001), polio 3 (p= 0.03) and Spn 23F (p=0.01) but not polio 1,2, PRP or Spn 6B, and 14 were decreased in sOP versus non-sOP children using generalized estimating equations. A high percentage of sOP children had non-protective antibody values that persisted until 24 months of age despite routine boosters.
sOP children may fail to achieve protective antibody concentrations after several routine vaccinations.
PMCID: PMC3884640  PMID: 24141796
vaccination; immunity; acute otitis media; pertussis; polio; hepatitis B; pneumococcal conjugate vaccine; dipththeria; tetanus; Haemophilus influenzae type b vaccine; immune deficiency
5.  Cellular Immune Response in Young Children Accounts for Recurrent Acute Otitis Media 
Current allergy and asthma reports  2013;13(5):10.1007/s11882-013-0370-z.
Acute otitis media (AOM) is a common disease in young children. Streptococcus pneumoniae(Spn) and Haemophilus influenzae (NTHi) are the two most common pathogens that cause AOM. Over the past 5 years our group has been studying the immunologic profile of children that experience repeated AOM infections despite tympanocentesis drainage of middle ear fluid and individualized antibiotic treatment; we call these children stringently-defined otitis-prone (sOP). Although protection against AOM is primarily mediated by ototpathogen-specific antibody, our recent studies suggest that suboptimal memory B-& T- cell responses and an immaturity in antigen presenting cells may play a significant role in the propensity to recurrent AOM infections. This review focuses on the studies performed to define immunologic dysfunction in sOP children.
PMCID: PMC3884676  PMID: 24022464
Acute otitis media; Cellular immune response; Recurrent otitis media; Streptococcus pneumoniae; Haemophilus influenzae; Memory T cells; Memory B cells; Cytokine response; Dendritic cells
6.  Acute Otitis Media Otopathogens during 2008-2010 in Rochester NY 
The otopathogen distribution colonizing the nasopharynx (NP) and causing acute otitis media (AOM) is in flux following the introduction of PCV7 and will continue to change.
277 children were followed prospectively; tympanocentesis was performed during AOM and 208 NP samples were collected to compare with middle ear fluid (MEF) isolates. 863 NP samples were collected at 7 healthy visits between 6 and 30 months of age. All children received PCV7 until April 2010 when PCV13 was substituted. Multi-locus sequence typing (MLST) was used to speciate S. pneumoniae.
The distribution of otopathogens in the MEF during the study time frame was stable. PCV7 serotypes of pneumococci were virtually absent. The frequency of isolation of S. pneumoniae was 26-36% compared with 28-34% for non-typeable Haemophilus influenzae (NTHi). M. catarrhalis isolation was less common, 7-18%. The proportion of S. pneumoniae that were penicillin non-susceptible was stable during the 3-years, 40-52%. All M. catarrhalis and 34% of NTHi were beta-lactamase-producing. NP isolates of otopathogens at onset of AOM included the isolate from the MEF and was dissimilar from the distribution at times of health. Sequence Types 320 and 199 of S. pneumoniae expressing serotypes 19A and 15 most often caused AOM.
The otopathogen distribution, antibiotic susceptibility and the diversity of strains within the S. pneumoniae species during 2008 through late 2010 were stable. NP isolation of otopathogens at onset of AOM better reflected, albeit incompletely, likely MEF isolates compared with NP isolates at times of health.
PMCID: PMC3755474  PMID: 23860479
acute otitis media; Haemophilus influenzae; Moraxella catarrhalis; Multilocus sequence typing; pneumococcal conjugate vaccine; Streptococcus pneumoniae
7.  Lower Nasopharyngeal Epithelial Cell Repair and Diminished Innate Inflammation Responses Contribute to the Onset of Acute Otitis Media in Otitis-Prone Children 
Medical microbiology and immunology  2013;202(4):295-302.
About 30% of young children experience excessive, frequent episodes of middle ear infection and are classified as acute otitis media prone (OP). Streptococcus pneumoniae (Spn) is a predominant otopathogen in OP and non-OP (NOP) children. The pathogenesis of middle ear infection involves otopathogen nasopharyngeal (NP) colonization followed by an upper respiratory viral infection that modifies the NP environment to allow a sufficient inoculum of bacteria to reflux via the Eustachian tube into the middle ear space. Here, we analyzed the NP mucosal repair response between age-matched OP and NOP children who progressed to middle ear infection caused by Spn. We found lower epidermal growth factor, epidermal growth factor receptor, and angiogenin cytokine concentrations in nasal washes of OP compared to NOP children. Despite higher expression of TLR2/4 transcript expression in nasal epithelium and in polymorphonuclear cells present in nasal secretions in OP children, OP children had lower expression of proinflammatory cytokines such as IL-6 and IL-8 in the NP. Chemotaxis associated cytokine expression at onset of AOM in OP children was also lower compared to NOP children, possibly indicating a lower capacity to signal the innate immune system. We conclude that lower epithelial cell repair responses during viral infection in the NP combined with diminished innate inflammatory responses potentiate Spn pathogenesis in the OP child.
PMCID: PMC3943207  PMID: 23576001
Streptococcus pneumoniae; Nasal Epithelial Repair; Innate immune responses; Acute Otitis Media; Otitis-prone
8.  CD4+ T-cell Responses Among Adults and Young Children In Response to Streptococcus pneumoniae and Haemophilus influenzae Vaccine Candidate Protein Antigens 
Vaccine  2013;31(30):3090-3097.
We characterized cytokine profiles of CD4+ T-helper (h) cells in adults and young children to ascertain if responses occur to next-generation candidate vaccine antigens PspA, PcpA, PhtD, PhtE, Ply, LytB of Streptococcus pneumonia (Spn) and Protein D and OMP26 of non-typeable Haemophilus influenzae (NTHi). Adults had vaccine antigen-specific Th1 - and Th2 cells responsive to all antigens evaluated whereas young children had significant numbers of vaccine antigen-specific CD4+ T cells producing IL-2, (p=0.004). Vaccine antigen-specific CD4+ T-cell populations in adults were largely of effector (TEM) and/or central memory (TCM) phenotypes as defined by CD45RA−CCR7+ or CD45RA−CCR7− respectively; however among young children antigen-specific IL-2 producing CD4+ T cells demonstrated CD45RA+ expression (non-memory cells). We conclude that adults have circulating memory CD4+T cells (CD45RA−) that can be stimulated by all the tested Spn and NTHi protein vaccine candidate antigens, whereas young children have a more limited response.
PMCID: PMC3777711  PMID: 23632305
Streptococcus pneumonia; non-typeable Haemophilus influenzae; CD4+ T-helper (h) cell; IFN-y; IL-2; memory T cells
9.  Reducing the Frequency of Acute Otitis Media by Individualized Care 
We sought to determine if use of more stringent diagnostic criteria for acute otitis media (AOM) than currently advocated by the American Academy of Pediatrics (AAP), tympanocentesis and pathogen-specific antibiotic treatment (individualized care) would result in reducing the incidence of recurrent AOM and consequent tympanostomy tube surgery.
A 5 year longitudinal, prospective study in Rochester NY was conducted from July 2006 – July 2011 involving 254 individualized care children. When this individualized care group developed symptoms of AOM, strict diagnostic criteria were applied and a tympanocentesis was performed. Pathogen resistance to empiric high dose amoxicillin/clavulanate (80mg/kg of amoxicillin component) caused a change in antibiotic to an optimized choice. Legacy controls (n=208) were diagnosed with the same diagnostic criteria by the same physicians as the individualized care group and received the same empiric amoxicillin/clavulanate (80mg/kg of Amoxicillin component) but no tympanocentesis or change in antibiotic. Community control children (n=1020) were diagnosed according to current AAP guidelines and treated with high dose amoxicillin (80 mg/kg) without tympanocentesis as guideline recommended.
5.9% of children of the individualized care group compared to 14.4% of Legacy controls and 27.3% of community controls became otitis prone (OP), defined as 3 episodes of AOM within a 6-month time span or 4 AOM episodes within a 12-month time span (p<0.0001). 2.4% of the individualized care group compared to 6.3% of Legacy controls, and 14.8% of community controls received tympanostomy tubes (p<0.0001).
Individualized care of AOM significantly reduces the frequency of AOM and tympanostomy tube surgery. Use of strict diagnostic criteria for AOM and empiric antibiotic treatment using evidence-based knowledge of circulating otopathogens and their antimicrobial susceptibility profile also produces improved outcomes.
PMCID: PMC3681924  PMID: 23340550
otitis prone; acute otitis media; tympanostomy tubes; tympanocentesis; amoxicillin; amoxicillin/clavulanate
10.  Higher serum levels of interleukin 10 occur at onset of acute otitis media caused by Streptococcus pneumoniae compared to Haemophilus influenzae and Moraxella catarrhalis 
The Laryngoscope  2013;123(6):1500-1505.
Acute otitis media (AOM) involves an inflammatory response to microbes in the middle ear that facilitates clearance of otopathogens. Clinically, Streptococcus pneumoniae (Spn) infections of the respiratory tract are characterized by greater inflammatory responses than nontypeable Haemophilus influenzae (NTHi) and Moraxella catarrhalis (Mcat). Interleukin 10 (IL-10) plays an important role in down-regulating the inflammatory response. We compared serum IL-10 levels in children before onset, at onset and after recovery from AOM caused by Spn, NTHi, and Mcat. We sought to determine if IL-10 could serve as a biomarker to distinguish AOM caused by Spn versus NTHi and Mcat.
Study Design
Prospective, longitudinal study in a primary care pediatric practice in Rochester, NY.
Participants were 54 children 6 to 30 months of age. Outcomes measured were serum IL-10 levels when healthy, at onset of AOM and after recovery from AOM.
Serum IL-10 was elevated when children developed AOM (p=0.013) due to infections caused by Spn (p= 0.011) but not AOM caused by NTHi or Mcat. Middle ear fluid levels of IL-10 mirrored those seen in serum but were 10-fold higher (p=0.02). Other effector cytokines in serum: IL-4, IFN-γ and TNF-alpha, did not show the same increases as IL-10 at onset of AOM.
Our study indicates that AOM caused by Spn elicits a significantly higher IL-10 response compared to NTHi and Mcat and may prove to be a biomarker of AOM infections by Spn.
PMCID: PMC3966296  PMID: 23404508
Interleukin 10; acute otitis media; Streptococcus pneumoniae; Haemophilus influenzae; Moraxella catarrhalis; cytokines
11.  Comparison of Amoxicillin/Clavulanate High Dose to Cefdinir in Treatment of Acute Otitis Media 
Drugs  2012;72(15):1991-1997.
To compare the clinical efficacy of amoxicillin/clavulanate high dose (Amox/clav HD) as 10 days therapy to cefdinir as 5 days therapy for acute otitis media (AOM).
Study Design
Diagnosis of AOM was based on specific criteria by validated otoscopists at 2 AOM research centers. The outcome measure was resolution of all symptoms and signs of AOM except persistence of middle ear effusion at test-of-cure (TOC) 12-15 days after antibiotic treatment .
330 children (x = 13.1 months) with AOM were studied. At TOC 256 children were cured, 69 failed and 5 were lost to follow-up. Amox/clav HD-treated children had a better cure rate (86.5%) than cefdinir (71.0%), p=0.001. Clinical outcomes showed that amox/clav HD was correlated with more frequent cure outcomes and that cefdinir was correlated with less frequent cure outcomes as children increased in age between 6 and 24 months.
When children have bona fide AOM an assessment of outcome is judged by validated otoscopists. 10 days amox/Clav HD is significantly more effective than 5 days of cefdinir as therapy for AOM. A trial comparing 10 days of cefdinir may have led to a different conclusion.
PMCID: PMC3963277  PMID: 23039319
Acute otitis media; Scoring systems; amoxicillin; cefdinir; antibiotics; clinical trial
12.  Antibody Response to Haemophilus influenzae Outer Membrane Protein D, P6, and OMP26 After Nasopharyngeal Colonization and Acute Otitis Media in Children 
Vaccine  2010;28(44):7184-7192.
Development of natural antibodies to 3 nontypeable Haemophilus influenzae (NTHi) outer membrane proteins (D, P6 and OMP26) was prospectively studied in 168 children 6–30 months of age during NP colonization and AOM. IgG antibody to protein D, P6 and OMP26 increased with age (p<0.001). Serum IgG responses to NP colonization were different for the 3 proteins: protein D responses occurred at a later age than P6, and OMP26 responses were minimal. For all 3 proteins serum antibody levels in the convalescent phase of AOM infection were not as high as after NP colonization. Antibodies to protein D and P6 but not OMP26 were bactericidal.
PMCID: PMC3959881  PMID: 20800701
Non-typeable Haemophilus influenzae; P6 protein; protein D; protein OMP26; Acute otitis media; carriage
13.  Nontypeable Streptococcus pneumoniae as an Otopathogen 
Among 34 Spn sequential isolates from middle ear fluid we found a case of a nontypeable Streptococcus pneumoniae (NT-Spn) in a child with AOM. The strain was pneumolysin PCR positive and capsule gene PCR negative. Virulence of the NT-Spn was confirmed in a chinchilla model of AOM.
PMCID: PMC3897191  PMID: 21251566
multi-locus sequence typing; nontypeable Streptococcus pneumoniae; acute otitis media
14.  Correlation of nasopharyngeal cultures prior to and at onset of acute otitis media with middle ear fluid cultures 
BMC Infectious Diseases  2014;14(1):640.
We sought to determine if nasopharyngeal (NP) cultures taken at times of healthy visits or at onset of acute otitis media (AOM) could predict the otopathogen mix and antibiotic-susceptibility of middle ear isolates as determined by middle ear fluid (MEF) cultures obtained by tympanocentesis.
During a 7-year-prospective study of 619 children from Jun 2006-Aug 2013, NP cultures were obtained from 6-30 month olds at healthy visits and NP and MEF (by tympanocentesis) at onset of AOM episodes.
2601 NP and 530 MEF samples were collected. During healthy visits, S. pneumoniae (Spn) was isolated from 656 (31.7%) NP cultures compared to 253 (12.2%) for Nontypeable Haemophilus influenzae (NTHi) and 723 (34.9%) for Moraxella catarrhalis (Mcat). At onset of AOM 256 (48.3%) of 530 NP samples were culture positive for Spn, 223 (42%) for NTHi and 251 (47.4%) for Mcat, alone or in combinations. At 530 AOM visits, Spn was isolated from 152 (28.7%) of MEF compared to 196 (37.0%) for NTHi and 104 (19.6%) for Mcat. NP cultures collected at onset of AOM but not when children were healthy had predictive value for epidemiologic antibiotic susceptibility pattern assessments.
NP cultures at onset of AOM more closely correlate with otopathogen mix than NP cultures at healthy visits using MEF culture as the gold standard, but the correlation was too low to allow NP cultures to be recommended as a substitute for MEF culture. For epidemiology purposes, antibiotic susceptibility of MEF isolates can be predicted by NP culture results when samples are collected at onset of AOM.
PMCID: PMC4264249  PMID: 25475135
Acute otitis media; Tympanocentesis; Streptococcus pneumoniae; Haemophilus influenzae (non-typeable); Moraxella catarrhalis
15.  Immunogenicity of Haemophilus influenzae Type b Protein Conjugate Vaccines in Very Low Birth Weight Infants 
PMCID: PMC3960569  PMID: 24569312
Infant; premature; infant; very low birth weight; Haemophilus influenzae vacines; immunization; vaccines
16.  PcpA of Streptococcus pneumoniae mediates adherence to nasopharyngeal and lung epithelial cells and elicits functional antibodies in humans 
Streptococcus pneumoniae (pneumococci) adhere to human nasopharyngeal (NP) epithelial cells as a first step in pathogenesis and adherence of pneumococci to lung epithelia may be required to establish pneumonia. We sought to determine if PcpA can serve as an adhesin to human NP (D562) and lung (A549) epithelial cells and whether PcpA mediated adherence can be inhibited by human anti-PcpA antibodies. A PcpA isogenic mutant was prepared on a wild type pneumococcal TIGR4 background. When the mutant and wild type strains were compared for adherence to D562 and A549 cell lines a reduction in adherence by the mutant was observed (p= 0.0001 for both cell types). PcpA was ectopically expressed on the surface of minimally-adherent heterologous host E coli resulting in augmented adherence to D562 (p= 0.002) and A549 (p= 0.015) cells. Total IgG was purified from a pool of 6 human sera having high IgG titers of anti-pneumococcal proteins. The purified IgG reduced TIGR4 adherence to D562 cells but we determined that this effect was largely due to bacterial cell aggregation as determined by flow cytometry and confocal microscopy. Fab fragments were prepared from pooled IgG sera. Inhibition of TIGR4 adherence to D562 cells was observed using the Fab fragments without causing bacterial aggregation (p=0.0001). Depletion of PcpA-specific Fab fragments resulted in an increase in adherence of TIGR4 to D562 cells (p=0.028). We conclude that PcpA can mediate adherence of pneumococci to human NP and lung epithelial cells and PcpA mediated adherence can be inhibited by human anti-PcpA antibodies.
PMCID: PMC3490615  PMID: 22796387
17.  Antibody response to Streptococcus pneumoniae proteins PhtD, LytB, PcpA, PhtE and Ply after nasopharyngeal colonization and acute otitis media in children 
We prospectively compared serum antibody levels of 5 Streptococcus pneumoniae (Spn) proteins: PcpA PhtD, PhtE Ply and LytB associated with nasopharyngeal (NP) colonization and acute otitis media (AOM) infection in a cohort of 6–30 mo old children. Antigen-specific antibody titers were determined by ELISA. A total of 731 visits among 168 children were studied. There were 301 Spn NP colonization episodes documented in 109 (65%) children and 42 Spn AOM episodes in 34 (20%) children. IgG antibody titers to the 5 proteins were significantly different among children over time (p < 0.001), with a rank order as follows: PcpA > PhtE = PhtD > Ply > LytB Characterization of IgG and IgM acute and convalescent serum antibody levels of Spn AOM infection showed the kinetics of the response differed among children, with the same rank order of antibody levels over time. Individual data showed that some children responded to AOM with an antibody increase to one or more of these Spn proteins but some children failed to respond. We conclude that antibody levels to Spn proteins PcpA PhtD, PhtE, Ply and LytB, all rise over time in children age 6 to 30 mo following natural exposure to Spn after NP colonization and AOM; however, there were significant differences in quantity of antibody elicited among these potential vaccine antigens.
PMCID: PMC3679215  PMID: 22495112
Acute otitis media; LytB; PcpA; PhtD; PhtE; Ply; Streptococcus pneumoniae
18.  Vaccine Candidates PhtD and PhtE of Streptococcus pneumoniae are adhesins that elicit functional antibodies in humans 
Vaccine  2012;30(18):2900-2907.
We evaluated the role of vaccine candidate surface proteins, PhtD and PhtE as antigens with functional importance for Streptococcus pneumoniae (pneumococci) in adherence to nasopharyngeal (D562) and lung (A549) epithelial cell lines. Comparing TIGR4 to PhtD and PhtE-deleted isogenic mutants, a 40% (p=0.001) and 42% (p=0.002) drop in the number of epithelial cells with adherent pneumococci was observed to both cells lines with the mutants, as quantitated using flow cytometry. We expressed PhtD and PhtE on the surface of E coli and demonstrated that when PhtD and PhtE were surface expressed on E coli adherence increased to D562 and A549 cells, compared with the E coli parent strain (p = 0.005, 0.013 for D562 and p=0.034, p=0.035 for A549). Using flow cytometry and confocal microscopy we found that pneumococci aggregated in the presence of human serum IgG, leading to a non-specific drop in adherence. Therefore IgG Fab fragments were prepared to study the functional role of PhtD and PhtE-specific Fabs in blocking adherence. The addition of 1 μg of IgG Fab from adult sera led to a 34% reduction (p= 0.002) and from children a 20% (p= 0.023) reduction in D562 epithelial cells with adherent pneumococci. In purified IgG from adult sera, the depletion of PhtD and PhtE specific Fab from total IgG Fab resulted in a significant increase in the number of D562 epithelial cells with adherent pneumococci (p=0.005 for PhtD and p=0.024 for PhtE). We conclude that antibody directed to PhtD and PhtE are adhesins of pneumococci, if raised by vaccination, may function to prevent pneumococcal adherence to human airway epithelial cells.
PMCID: PMC3490617  PMID: 22349524
19.  Reduced Serum IgG Responses to Pneumococcal Antigens in Otitis-Prone Children May Be Due to Poor Memory B-Cell Generation 
The Journal of Infectious Diseases  2012;205(8):1225-1229.
A low level of serum antibody to antigens expressed by Streptococcus pneumoniae has been proposed to explain the susceptibility of children to recurrent episodes of acute otitis media (hereafter, “otitis-prone children”). By use of enzyme-linked immunospot assays, the percentages of memory B cells to pneumococcal protein antigens PhtD, LytB, PcpA, PhtE, and Ply were compared between otitis-prone and non–otitis-prone children at the time of acute otitis media or nasopharyngeal colonization with S. pneumoniae. We found significantly lower percentages of memory B cells to 3 pneumococcal protein antigens (PhtD, PhtE, and Ply) and reduced antigen-specific immunoglobulin G concentrations in otitis-prone children, compared with non–otitis-prone children.
PMCID: PMC3308907  PMID: 22383675
20.  Antibody in Middle Ear Fluid of Children Originates Predominantly from Sera and Nasopharyngeal Secretions 
Clinical and Vaccine Immunology : CVI  2012;19(10):1593-1596.
The human middle ear is devoid of any immunocompetent cells in normal mucosa. We sought to determine the source of antibody present in the middle ear of children. Total IgG, IgA, and secretory IgA antibodies were determined by enzyme-linked immunosorbent assay from the nasopharyngeal, middle ear, and serum samples of children with acute otitis media. The two-dimensional gel electrophoresis pattern of the entire array of IgA antibodies in the nasal wash (NW) and middle ear fluid (MEF) was compared from the MEF and NW samples using isoelectric focusing and Western blotting. The total IgG and IgA antibodies in the MEF and NW samples of 137 children were compared. The ratio of IgG to IgA in the MEF was significantly different (P < 0.008) compared to NW because IgA levels were higher and IgG levels lower in NW. The IgG/IgA ratio of MEF resembled serum consistent with transudation to the MEF. Small amounts of secretory IgA were detected in MEF but the electrophoresis patterns of the entire array of IgA antibodies in the MEF and NW were virtually identical in each child evaluated; thus, IgA in MEF derived predominantly from serum and the nasopharynx by reflux via the Eustachian tube. The IgG/IgA antibody levels in the MEF and the same composition of IgA antibody in the MEF and NW identifies the predominant source of antibody in the MEF as a transudate of serum combined with nasal secretions refluxed from the nasopharynx in children.
PMCID: PMC3485881  PMID: 22855395
21.  Clinical Significance of Serum S100A12 in Acute Otitis Media in Young Children 
S100A12 is a calcium-binding protein predominantly expressed in neutrophil granulocytes in response to infections or inflammation. Acute otitis media (AOM) is a local infection mainly caused by Streptococcus pneumoniae (Spn), nontypeable Haemophilus influenzae (NTHi) and/or Moraxella catarrhalis (Mcat).
To study if S100A12 values could serve as a diagnostic marker, serum S100A12 concentrations were tested in young children before, at onset and after recovery from AOM.
Among 116 children with AOM we found that serum S100A12 concentrations were significantly increased at onset of AOM compared to before infection (p=0.0001), and returned to normal levels when the children recovered from the infection (p=0.01). Elevation of S100A12 correlated with the presence of Spn (p=0.004) or NTHi (p=0.04) in the middle ear, but not with Mcat or upper respiratory viral infection. Change in serum value of S100A12 at onset of AOM were not related to the frequency of occurrence of AOM or the age of the child.
S100A12 may be a useful biomarker for onset of AOM due to Spn and NTHi; and for following children with AOM.
PMCID: PMC3308144  PMID: 22198825
S100A12; acute otitis media; biomarker; young children
22.  Simultaneous Assay for Four Bacterial Species Including Alloiococcus otitidis Using Multiplex-PCR in Children With Culture Negative Acute Otitis Media 
The 3 most commonly encountered bacteria in acute otitis media (AOM) are Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Conventional culture methods detect these pathogens in only 60% to 70% of cases of AOM. Alloiococcus otitidis, another potential pathogen, has often been ignored.
Tympanocentesis was performed in 97 children with AOM presenting with a bulging tympanic membrane (TM) producing 170 middle ear fluids (MEFs). S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis were isolated in 21%, 32%, 8%, and 0% of MEFs, respectively; no otopathogen was isolated in 29% of MEFs. In nasopharyngeal cultures at the time of AOM diagnosis, 34%, 36%, 17%, and 0% and in oropharyngeal cultures, 7%, 31%, 11%, and 0% grew S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis, respectively. No otopathogen was isolated in 23% of nasopharyngeal and 20% of oropharyngeal cultures. Multiplex polymerase chain reaction (PCR) was used to detect DNA of the 4 bacterial species in culture negative samples.
All culture-positive MEF, nasopharyngeal and oropharyngeal samples tested were also multiplex-PCR positive, indicating the reliability of the method. Culture-negative samples of MEF from children with a bulging TM yielded S. pneumoniae, H. influenzae, M. catarrhalis, and A. otitidis DNA in 51%, 35%, 14%, and 32% of MEF, in 45%, 31%, 10%, and 9% of nasopharyngeal and in 31%, 23%, 0%, and 3% of oropharyngeal, respectively. In 9% of the cases A. otitidis DNA was found without detection of a second organism in MEF.
Conventional culture detected otopathogens in MEF of children with a bulging TM in 71%; using multiplex-PCR, otopathogens were detected in 88% of MEF (P < 0.01). Similar improved detection of otopathogens was noted with nasopharyngeal and oropharyngeal cultures.
PMCID: PMC3581301  PMID: 20335823
acute otitis media; Alloiococcus otitidis; multiplex-PCR; otopathogen; Streptococcus pneumoniae
23.  Phylogenetic relatedness and diversity of non-typable Haemophilus influenzae in the nasopharynx and middle ear fluid of children with acute otitis media 
Journal of Medical Microbiology  2011;60(Pt 12):1841-1848.
The phylogenetic relationships of non-typable Haemophilus influenzae (NTHi) strains prospectively isolated from healthy children and children with acute otitis media (AOM) were analysed using multilocus sequence typing (MLST). A total of 165 NTHi isolates were collected over a 3.5 year time frame during 2006 through 2009. The strains were tested for β-lactamase production; 28.5 % were positive. Seventy different NTHi sequence types (STs) were identified of which 29 (41.4 %) were novel. NTHi strains did not show any phylogenetic grouping or clustering among asymptomatic colonizing strains or strains that caused AOM, or based on β-lactamase enzyme production. Evaluation of triplets and other siblings over time demonstrated relatively frequent genetic exchanges in NTHi isolates in vivo in a short time frame and subsequent transfer among children in a family. Comparison of the MLST STs isolated at different time points showed that in ~85 % of the nasopharynx (NP) colonizations, NTHi strains cleared from the host within 3 months, that sequential colonization in the same child involved different strains in all cases except one, and that NP and middle ear isolates were identical STs in 84 % of cases. In this first study of its type to our knowledge, we could not identify predominant MLST types among strains colonizing the NP versus those causing AOM or expressing a β-lactamase enzyme conferring penicillin resistance in children.
PMCID: PMC3347915  PMID: 21799196
24.  Transcriptome signature in young children with acute otitis media due to non-typeable Haemophilus influenzae  
International Immunology  2013;25(6):353-361.
The transcriptome during acute otitis media due to NTHi shows relatively weak immune activation
Non-typeable Haemophilus influenzae (NTHi) causes acute otitis media (AOM) in young children. In our recent paper in Microbes and Infection we described the transcriptome signature elicited from PBMCs at onset of AOM caused by Streptococcus pneumoniae. In the current study we found very different results with NTHi AOM infections; 5.1% of 29 187 genes were differentially regulated by more than 2-fold at the onset of AOM compared with the pre-infection healthy state in the same children. Among the 1487 transcripts, 100 genes associated with the immune defense response were specifically analyzed. About half of the differentially regulated genes associated with antibacterial activity and the cell-mediated immune response were activated and half were suppressed. The important signatures for NTHi in children suggested that the balance of the immune response was toward suppression. Moreover, 90% of the genes associated with a pro-inflammatory cytokine response were down-regulated. The genes associated with the classic complement pathway were down-regulated, although the alternative complement pathway genes were up-regulated. These results provide the first human transcriptome data identifying gene expression in the immune response to be predominantly down-regulated at the onset of AOM due to NTHi.
PMCID: PMC3708516  PMID: 23411728
acute otitis media; immune response signatures; non-typeable Haemophilus influenzae; transcriptome profile
25.  Reduced Memory CD4+ T-Cell Generation in the Circulation of Young Children May Contribute to the Otitis-Prone Condition 
The Journal of Infectious Diseases  2011;204(4):645-653.
Background. An explanation for the immunologic dysfunction that causes children to be prone to repeated episodes of acute otitis media (AOM) has long been sought. Poor antibody response has been associated with the otitis-prone condition; however, there is no precise mechanistic explanation for this condition.
Methods. Non–otitis-prone and otitis-prone children with AOM or nasopharyngeal (NP) colonization caused by either Streptococcus pneumoniae or Haemophilus influenzae were compared for pathogen-specific CD4+ T-helper memory responses by stimulating peripheral blood mononuclear cells using 6 vaccine candidate S. pneumoniae and 3 H. influenzae protein antigens. Samples were analyzed by multi-parameter flow cytometry.
Results. Significantly reduced percentages of functional CD45RALow memory CD4+ T cells producing specific cytokines (interferon γ, interleukin [IL]–2, IL-4 and IL-17a) were observed in otitis-prone children following AOM and NP colonization with either S. pneumoniae or H. influenzae. Immunoglobulin (Ig) G responses to the studied protein antigens were reduced, which suggests that antigen-specific B-cell function may be compromised as a result of poor T-cell help. Staphylococcal enterotoxin B stimulated similar cytokine patterns in memory CD4+T cells in both groups of children.
Conclusions. Otitis-prone children have suboptimal circulating functional T-helper memory and reduced IgG responses to S. pneumoniae or H. influenzae after colonization and after AOM; this immune dysfunction causes susceptibility to recurrent AOM infections.
PMCID: PMC3144174  PMID: 21791667

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